Ra p id On e-P ot Syn th esis of Ribofla vin Isotop om er s
Werner R o¨ misch, Wolfgang Eisenreich, Gerald Richter,* and Adelbert Bacher
Lehrstuhl f u¨ r Organische Chemie und Biochemie, Technische Universit a¨ t M u¨ nchen, Lichtenbergstr. 4,
D-85747 Garching, Germany
Received J une 26, 2002
Flavocoenzymes labeled with stable isotopes are important reagents for the study of flavoproteins
using isotope-sensitive methods such as NMR, ENDOR, infrared, and Raman spectroscopy. We
describe highly versatile one-pot methods for the preparation of riboflavin isotopomers labeled with
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C in every desired position of the xylene moiety. The starting materials are commercially available
C-labeled glucose samples, which are converted into riboflavin using enzymes of the oxidative
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pentose phosphate pathway in combination with recombinant enzymes of the riboflavin biosynthetic
pathway. The overall reaction comprises six enzyme-catalyzed reaction steps for the synthesis of
the vitamin and two auxiliary enzymes for in situ recycling of cofactors. The overall yields of
riboflavin based on isotope-labeled glucose are 35-50%.
In tr od u ction
Raman spectroscopy of flavoproteins and can contribute
substantially to electron spin resonance experiments
using nuclear spin sensitive methods such as ENDOR.
Ideally, these spectroscopic techniques should utilize
protein samples carrying a variety of differently isotope-
substituted flavocoenzymes. Synthetic procedures for a
Flavocoenzymes derived from the vitamin riboflavin (8)
are absolutely required in all organisms, and their central
role in redox biochemistry has been investigated in
considerable detail.1 Flavins are also involved in a
variety of nonredox processes such as blue light reception
13-20
-5
number of riboflavin isotopomers have been reported in
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7
in plants, photorepair of photodamaged DNA, and in
10,21,22
the literature,
but the preparation of a reasonably
circadian time-keeping.8
comprehensive library of isotopomers is laborious and
time-consuming.
A more elegant approach to the preparation of isoto-
pomer libraries by parallel synthesis is possible by
enzyme-assisted procedures. The enzymes of the ribofla-
The properties of the flavin chromophore can be
modulated over a wide range by the protein environment
in a specific flavoprotein. Monitoring the state of the
flavin chromophore by physical techniques is therefore
crucial for the mechanistic analysis of flavoproteins.
vin biosynthetic pathway can be expressed in high yields
in recombinant microbial hosts.2
3-26
As shown in this
NMR spectroscopy has been used extensively for
mechanistic studies of flavoproteins.
1
,9-12
This approach
requires stable isotope labeling of the flavin cofactor in
order to enhance the selectivity and sensitivity of NMR
detection. Stable isotope labeling of flavin cofactors can
also serve as the basis for time-resolved infrared and
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*
Corresponding author. Tel. +49-89-289-13336. Fax: +49-89-289-
3363.
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0.1021/jo026105x CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/09/2002
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J . Org. Chem. 2002, 67, 8890-8894